The repair of Double Strand Breaks (DSB) in DNA is essential for the maintenance of genomic integrity. A growing body of evidence identifies the Non-Homologous End-Joining (NHEJ) mechanism of DSB repair as central to genome maintenance and suggests that deficiencies in the NHEJ apparatus can generate oncogenic events that drive general tumorigenesis. Genetic research has identified many of the key factors that function in mammalian NHEJ. Among these the Ku protein is thought to play an important, but as yet uncharacterized, role. The previous discovery that an inositol phosphate (a small, highly phosphorylated molecule) is bound by Ku and stimulates NHEJ in mammalian cell extracts provides us with a logical starting point from which we plan to investigate the mechanism of this process. In this proposal the roles of Ku and inositol phosphate in the NHEJ reaction will be examined in vitro and in vivo. Despite our understanding of the factors that participate in NHEJ, this process has not yet been reconstituted in vitro. Additionally, a new cell line that is deficient in NHEJ (but normal for all known NHEJ factors) has been discovered. Taken together these data suggests that additional, as yet unidentified, factors may play a role in this process. In addition to our work in characterizing the role of Ku and inositol phosphate we propose to identify additional participants in the mammalian NHEJ reaction. My long-term research aims target the molecular mechanism of DSB repair by NHEJ in mammalian cells. An understanding of this system will provide insight into a mechanism that appears to drive tumor formation and may identify a means of controlling NHEJ in mammalian cells to provide an effective cancer treatment.